methods
Quantification of sterol lipids in plants by quadrupole
time-of-flight mass spectrometry
Vera Wewer, Isabel Dombrink, Katharina vom Dorp, and Peter Dörmann1
Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, 53115 Bonn, Germany
Abstract Glycerolipids, sphingolipids, and sterol lipids
constitute the major lipid classes in plants. Sterol lipids are
composed of free and conjugated sterols, i.e., sterol esters,
sterol glycosides, and acylated sterol glycosides. Sterol lip-
ids play crucial roles during adaption to abiotic stresses and
plant-pathogen interactions. Presently, no comprehensive
method for sterol lipid quantification in plants is available.
We used nanospray ionization quadrupole-time-of-flight
Q1
mass spectrometry (Q-TOF MS) to resolve and identify the
molecular species of all four sterol lipid classes from Arabi-
dopsis thaliana. Free sterols were derivatized with chloro-
betainyl chloride. Sterol esters, sterol glycosides, and acylated
sterol glycosides were ionized as ammonium adducts.
Quantification of molecular species was achieved in the
positive mode after fragmentation in the presence of inter-
nal standards. The amounts of sterol lipids quantified by
Q-TOF MS/MS were validated by comparison with results
obtained with TLC/GC. Quantification of sterol lipids from
leaves and roots of phosphate-deprived A. thaliana plants
revealed changes in the amounts and molecular species
composition. The Q-TOF method is far more sensitive than
GC or HPLC. Therefore, Q-TOF MS/MS provides a com-
prehensive strategy for sterol lipid quantification that can
be adapted to other tandem mass spectrometers.—Wewer,
V., I. Dombrink, K. vom Dorp, and P. Dörmann. Quantifica-
tion of sterol lipids in plants by quadrupole time-of-flight
mass spectrometry. J. Lipid Res. 2011. 52: 1039–1054.
Supplementary key words Arabidopsis • campesterol • cholesterol •
phosphate limitation • phytosterol • sitosterol • stigmasterol
Glycoglycerolipids (monogalactosyl diacylglycerol, diga-
lactosyl diacylglycerol [DGDG], and sulfolipid [SQDG])
are the most abundant membrane lipids in chloroplasts of
plants (1, 2). Different lipid classes, however, are prevalent
in extraplastidial membranes. Phosphoglycerolipids (e.g.,
phosphatidylcholine and phosphatidylethanolamine) are
abundant constituents of the plasma membrane, the to-
noplast, and the endoplasmic reticulum. Furthermore,
This work was supported by an Instrument grant (Forschungsgrossgeräte-
Antrag) and by the Forschungsschwerpunkt 1212 of Deutsche Forschungsgemein-
schaft (grant Do520/9).
Manuscript received 6 January 2011 and in revised form 14 February 2011.
Published, JLR Papers in Press, March 7, 2011
DOI 10.1194/jlr.D013987
Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
sphingolipids, in particular glucosylceramides and glyco-
sylinositolphosphoceramides, are present in extraplastid-
ial membranes of plants (3, 4). In addition to glycerolipids
and sphingolipids, sterol lipids represent the third mem-
brane lipid class in plants. In contrast to animals and yeast,
where cholesterol and ergosterol, respectively, are preva-
lent, phytosterols (campesterol, stigmasterol, and sitos-
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terol) represent the most abundant sterols in plants (5).
The biosynthetic pathway for phytosterols also provides
the precursors for brassinosteroids, phytohormones in-
volved in the regulation of plant growth and development
(6).
Free sterols (FS) with a nonconjugated 3-hydroxy group
Q3
are found in the plasma membrane and other extraplas-
tidial membranes of plant cells. In addition, different
classes of conjugated sterols are found in plants, sterol es-
ter (SE), sterol glucoside (SG), and acylated (esterified)
sterol glucoside (ASG). Plant cells contain considerable
amounts of SEs, which in contrast to the other sterol lipid
classes, are localized to the oil bodies of the cytosol (7, 8).
Oil bodies are the sites of lipid storage and contain large
amounts of triacylglycerol and SEs. Two acyltransferases
were identified in Arabidopsis, acyl-CoA:sterol acyltrans-
ferase and phospholipid:sterol acyltransferase (PSAT),
capable of transferring acyl groups from acyl-CoA or from
a phosphoglycerolipid, respectively, onto the 3-hydroxy
group of sterols (9, 10). A deficiency in PSAT activity
causes early leaf senescence, and it is believed that acyla-
tion of sterols is crucial for the regulation of the FS con-
tent in plant membranes (11).
SGs carry a D-glucose in b-glycosidic linkage of the 3-hy-
droxy group of the sterol (12, 13). The first gene encoding
sterol glucosyltransferase was isolated from oat (14). Two
Abbreviations: ASG, acylated sterol glucoside; DGDG, digalactosyl
diacylglycerol; DW, dry weight; FAG, acyl-glucoside-specific fragment;
FB, betainyl fragment; FS, sterol-specific fragments; FS, free sterol; FW,
fresh weight; Glc, glucose; MS/MS, tandem mass spectrometry; PSAT,
phospholipid-sterol acyltransferase; Q-TOF, quadrupole time-of flight;
SE, sterol ester; SG, sterol glucoside; SPE, solid phase extraction; SQDG,
sulfolipid; WT, wild type.
1
To whom correspondence should be addressed.
e-mail: doermann@uni-bonn.de
Journal of Lipid Research Volume 52, 2011 1039
genes encoding sterol glucosyltransferases are present in
Arabidopsis. A mutant line carrying insertions in the two
Arabidopsis genes is affected in seed coat formation and
suberization (15). Acylation with a long chain fatty acid at
position 6 of the glucose moiety results in the formation of
ASG (16). SGs and ASGs are found in the plasma mem-
brane of plants and some yeast species (17). Sterol lipids
are believed to contribute to diverse functions in the plant
cell. Different forms of sterols are involved in plant–
pathogen interactions. A recent study showed that in Arab-
idopsis, sitosterol is converted into stigmasterol during
Pseudomonas infection (18). Furthermore, sterol glucosides
play important roles during the immune response in humans
after Helicobacter infection and in peroxisome degradation
in Pichia (19, 20).
Recently, the role of phosphate availability for glycero-
lipid metabolism has been studied in great detail. Phos-
phate deprivation results in the replacement of a large
proportion of phosphoglycerolipids with DGDG and
SQDG to save phosphate for other important cellular pro-
cesses (2, 21). This alteration in membrane lipid composi-
tion is associated with an upregulation of the expression of
the biosynthetic genes for DGDG and SQDG, demonstrat-
ing that this adaptive process is regulated via activation of
gene expression. The role of other glycolipids during
phosphate deficiency, including SGs and ASGs, is less well
understood. ASGs were found to be particularly increased
in detergent-insoluble membranes of oat root plasma
membranes during phosphate deprivation (22). At pres-
ent, no data for the molecular species composition of SEs
and of ASGs during normal conditions or phosphate de-
privation are available.
Due to the presence of a large number of molecular
species, the measurement of the different sterol lipids in
plants is challenging. Previously, membrane lipids, includ-
ing sterols, were separated by HPLC via normal phase
chromatography coupled with evaporative light scattering
detection (23, 24). However, this approach is hampered by
a limitation in sensitivity and linearity of the detector
signal. Furthermore, no information on molecular species
composition can be obtained. An alternative approach in-
cludes the separation and isolation of different sterol lipid
classes via TLC and the quantification of the sterol moi-
eties by GC after hydrolyzation (11, 15, 22). This strategy
provides data on the sterol composition of sterol lipid clas-
ses but not on the composition of individual molecular spe-
cies of SEs and ASGs. Furthermore, two different TLC
systems are required to separate the polar (SG and ASG)
and the nonpolar sterol lipids (FS and SE). In addition,
this method is labor intensive and suffers from a lack of
sensitivity.
Recently, the availability of high-resolution mass spec-
trometers has resulted in the development of highly sen-
sitive and accurate methods for the identification and
quantification of lipids (25). Phosphoglycerolipids and
glycoglycerolipids can be measured in total leaf lipid ex-
tracts by direct infusion ESI coupled with triple quadru-
pole mass spectrometry (MS) (26, 27). In this strategy, the
masses of the individual molecular lipid species are se-
1040 Journal of Lipid Research Volume 52, 2011
lected by the first quadrupole, and, after fragmentation,
the lipids are quantified using characteristic fragmenta-
tion patterns (neutral loss or precursor ion scanning). The
separation of lipids via HPLC coupled with a quadrupole
ion trap (Q-trap) mass spectrometer was employed for the
quantification of plant sphingolipids (3, 4). After separa-
tion by HPLC, the sphingolipids are quantified via multiple
reaction monitoring, taking advantage of the characteristic
mass transitions after fragmentation. An alternative strat-
egy, i.e., direct infusion quadrupole time-of-flight MS (Q-
TOF MS) was applied to the quantitative analysis of yeast
lipids including phospholipids, sphingolipids, triacylglyc-
erol, and diacylglycerol (28). A number of methods were
recently developed for the analysis of cholesterol, oxy-
sterols, and related sterols from animals or humans by LC
or ESI-MS (29–33). Thus, free sterols were usually deriva-
tized to improve ionization, and derivatized FSs and SEs
were analyzed in tandem MS (MS/MS) experiments. How-
ever, for plants, no comprehensive method for sterol lipid
analysis was developed.
The goal of the present study was to establish a method
for the detection, identification, and quantification of the
Downloaded from www.jlr.org by guest, on December 9, 2020
different molecular species of all four sterol lipid classes in
plants by employing nanospray ionization Q-TOF MS/MS.
EXPERIMENTAL PROCEDURES
Plants and growth conditions
Arabidopsis thaliana, ecotype Columbia-0, was grown in pots in
a mixture of soil and vermiculite (2:1) for 4 weeks in phytotrons
22 21
at 20°C, 8 h of light (150 µmol m s ) per day, and 55% relative
humidity. For phosphate deprivation, seedlings were grown on
agar-solidified medium containing Murashige and Skoog salts for
2 weeks (34) and then grown on synthetic nutrient medium for
an additional 2 weeks (35, 36). The pho1-2 mutant (37, 38) was
Q4
obtained from the Nottingham Arabidopsis Seed Center (Notting-
ham, UK). The pho1-2 mutant (a loss-of function mutant allelic to
pho1-1) is abbreviated as pho1 throughout the text.
Lipid standards
Cholestanol and stigmastanol (Sigma) were used as internal
Q5
standards for the quantification of FSs. Internal standards for
the quantification of SEs were 16:0-cholesterol, 16:1-cholesterol,
18:0-cholesterol, and 18:1-cholesterol (fatty acids are abbreviated
X:Y, where X represents the number of carbon atoms, and Y rep-
resents the number of double bonds in the acyl chain) (Sigma).
The SGs used as internal standards were synthesized as described
previously (39) using cholestanol and stigmastanol. Briefly, 1.25 g
of glucopyranosyl-bromide-tetrabenzoate were dissolved in 10 ml
of toluene and added to a suspension of 0.61 g of sterol, 2 g of
drierite, and 2 g of CdCO3 in 12 ml of toluene. The mixture was
incubated at 130°C for 7 h. After cooling, the suspension was di-
luted with 24 ml of chloroform and filtered after celite was added.
The filtrate was dried under a stream of N2, resuspended in 40 ml
of 0.15 M sodium methylate, and incubated overnight with shak-
ing at room temperature. The solution was neutralized with 1 N
methanolic HCl, and the products were extracted with chloroform-
methanol (2:1) and water. The SGs were purified by solid phase
extraction (SPE) on silica columns (see below) or by TLC and
quantified by GC. Acylated (esterified) sterol glucosides from
soybean (containing stigmasterol, sitosterol, and campesterol;
Matreya, Pleasant Gap, PA) were hydrogenated with H2 gas in
chloroform in the presence of platinum(IV)oxide as described
previously (40). Complete saturation of the acyl and sterol moi-
eties was confirmed by Q-TOF MS/MS analysis. Standard sterol
lipids were hydrolyzed with methanolic HCl, and fatty acids were
converted into their methyl esters as described below. Fatty acid
methyl esters derived from SE and ASG were quantified by GC.
The sterol moieties of FS, SG, and ASG were converted into tri-
methylsilyl ethers and quantified by GC. The standard lipid mixture
(in 50 µl of chloroform-methanol, 2:1) contained 5 nmol each of
cholestanol and stigmastanol; 2.5 nmol each of 16:0-cholesterol,
18:0-cholesterol, 16:1-cholesterol, and 18:1-cholesterol; 5 nmol
each of cholestanol-Glc and stigmastanol-Glc; 0.2 nmol of 16:0-
Glc-campestanol; 0.5 nmol of 16:0-Glc-stigmastanol; 0.3 nmol of
18:0-Glc-campestanol; and 1 nmol of 18:0-Glc-stigmastanol.

Extraction of total lipids and SPE

Arabidopsis leaves or roots (20–100 mg fresh weight [FW]) were
ground to a fine powder in liquid N2. Total lipids were extracted
with 500 µl of chloroform-methanol-formic acid (1:1:0.1) and
250 µl of a solution of 1 M KCl and 0.2 M H3PO4. All organic
solvents contained 0.01% butylated hydroxytoluene as antioxi-
dant. The internal standard mixture (50 µl) was added. The sam-
ple was vortexed and centrifuged (7,500 g, 2 min) to obtain phase

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separation. The lower, organic phase was harvested with a pi-
pette. Two additional extractions with 250 µl of chloroform-
methanol (2:1) each were done, and the organic phases were
combined. The solvent was evaporated under a stream of N2 gas.
Nonpolar lipids, glycolipids, and phospholipids were separated
by SPE using Strata silica columns (1 ml bed volume; Phenome-
nex). Lipids were applied to the silica columns in 100% chloro-
form, and nonpolar lipids (including FS and SE) were eluted
with 2 ml of chloroform. The glycolipid fraction containing SG
and ASG was obtained by elution with 1 ml of acetone-2-propanol
(1:1).The purified lipid extracts containing FS/SE or SG/ASG
were dried and dissolved in 1 ml of Q-TOF solvent (see below).
FSs were derivatized with N-chlorobetainyl chloride prior to
Q-TOF analysis (see below).
Quantification of sterol lipids by TLC and GC
Lipids were extracted from leaves (2 g of material) and sepa-
rated into nonpolar and polar fractions by SPE on silica columns,
as described above. The nonpolar fraction containing FS and SE
and the polar fraction containing SG and ASG were each divided
into two aliquots. One aliquot was used for Q-TOF analysis, and
the other aliquot was used for quantification by TLC/SPE and
GC (Fig. 1). Nonpolar lipids (FS and SE) were separated by an
additional step of SPE with a silica column by application to the
column in 100% hexane and elution with hexane-diethylether
(99:1) (for SE) and hexane-diethylether (85:15) (for FS) (see
http://www.cyberlipid.org). For the TLC separation of polar lip-
ids, Baker Si 250 TLC plates (J.T. Baker, Phillipsburg, NJ) were
impregnated with 0.15 M ammonium sulfate and activated for
2.5 h at 120°C. The TLC plates were developed in acetone-
toluene-water (91:30:8) (41), and lipids were isolated from the
silica material with chloroform-methanol (2:1). For GC analysis, SEs
were hydrolyzed in 1 ml of 6% (w/v) KOH in methanol at 90°C
for 1 h and then extracted with hexane (42). SG and ASG were
hydrolyzed in 1 ml of 1 N methanolic HCl at 90°C for 30 min.
After the addition of 1 ml of 0.9% NaCl, the hydrolyzed sterols
were extracted with 1 ml of hexane. Sterols were derivatized with
100 µl of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA)
prior to GC analysis. MSTFA was evaporated in an N2 gas stream,
and the samples were dissolved in hexane. GC analysis was car-
ried out with an Agilent 7890A Plus GC unit with flame ioniza-
Fig. 1. Flow chart for sterol lipid quantification. Sterol lipids
were obtained from ⵑ2 g of leaf material and, after fractionation
by TLC and SPE, were quantified by GC. The direct infusion MS
strategy is based on lipid extracts obtained from ⵑ20 mg of leaf
material. Lipids are fractionated by SPE and quantified by Q-TOF
MS/MS.
tion detector (Agilent). Silylated sterols were separated on a 30 m
HP-5MS column (Agilent) using a temperature gradient of 150°C
increased to 280°C at 10°C min21, held for 10.5 min, and de-
creased to 150°C at 20°C min21. Sterols were identified by GC-MS
(Agilent) using the same column and temperature gradient.
Derivatization of free sterols for Q-TOF MS/MS
Free sterols were derivatized with N-chlorobetainyl chloride
following the procedure described for diacylglycerol derivatiza-
tion (43). Briefly, lipids (nonpolar fraction after SPE purifica-
tion) were dissolved in 0.5 ml of anhydrous methylene chloride.
Then, 50 µl of anhydrous pyridine and 5 mg of N-chlorobetainyl
chloride (see below) were added, and the sample was incubated
at 42°C for 4 h (or overnight with the same results). The betainy-
lated sterols were extracted with 2 vol of chloroform-methanol
(1:1) and 1 vol of water. Derivatized sterols were stable for at least
48 h at 18°C (as measured by Q-TOF MS/MS analysis). For the
synthesis of N-chlorobetainyl chloride, 1 g of betaine hydrochlo-
ride was added to 0.93 g of thionyl chloride (44). The mixture
was slowly heated to 70°C and incubated overnight. After the ad-
dition of 1 ml of warm (ⵑ70°C) toluene, the reaction mixture
was cooled to room temperature with stirring. More warm tolu-
ene was added, and the reaction mixture was heated until the
substances were dissolved. After cooling again, crystals formed,
which were washed twice with methylene chloride, dried, and
stored at 4°C.
Q-TOF MS
Sterols purified by SPE were introduced into the Q-TOF mass
spectrometer (Agilent 6530 Accurate Mass Q-TOF LC-MS unit)
operated in the positive mode via direct nanospray infusion

Sterol lipid quantification in plants by Q-TOF MS 1041

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Fig. 2. Mass spectra of sterol lipids without fragmentation. Sterol lipids were directly infused into the Q-TOF mass spectrometer via nano-
spray ionization. Ion spectra were recorded in the positive mode without fragmentation. A: Free sterols from Arabidopsis leaves after SPE
purification and derivatization as betainylchloride esters. Cholestanol and stigmastanol were used as internal standards. B: Sterol esters
from Arabidopsis leaves after two-step SPE purification. The following internal standards were included: 16:0-cholesterol, 16:1-cholesterol,
18:0-cholesterol, and 18:1-cholesterol. C: Sterol glucosides (soybean); internal standards were cholestanol-Glc and stigmastanol-Glc. D:
Acylated sterol glucosides (soybean); 16:0-Glc-stigmastanol, 18:0-Glc-stigmastanol, 16:0-Glc-campestanol, and 18:0-Glc-campestanol were
the internal standards.

(HPLC chip/MS 1200 with infusion chip; Agilent). Sterol lipids of the betainyl fragment, FB, of the extracted ion chromatogram
were supplied to the mass spectrometer in methanol-chloroform- function, which corresponds to the precursor ion scan function
300 mM ammonium acetate (665:300:35) (26) at a flow rate of 1 of a triple quadrupole instrument (see Fig. 3). Betainyl deriva-
µl min21. The temperature of the nitrogen gas in the collision tives of unsaturated sterols consistently resulted in lower MS/MS
cell was 300°C at a flow rate of 8 liters min21. The fragmentor signal intensities than derivatives of saturated sterols. For this rea-
voltage was 200 V, and the capillary voltage (Vcap) was set to son, a mixture of soybean sterols (campesterol, stigmasterol, and
1,700 V. MS spectra without fragmentation (“MS only” mode) sitosterol), cholestanol, and stigmastanol was quantified by GC
were recorded after every fifth MS/MS spectrum. For MS/MS
measurements, the quadrupole was operated in the narrow range TABLE 1. Profiling of free sterols from Arabidopsis
(m/z, 1.2) and was set at the exact mass of the sterol lipid. Q-TOF
Compound (M + betainyl)+ Product ion
MS/MS spectra were accumulated for 1 s, and the signal intensi- number
a
Sterol lipid Formula (M)b (m/z)
c
FB(m/z)
ties of at least five different spectra derived from the same paren-
tal ion were averaged. Collision energies for MS/MS spectra for 1 Cholesterol C27H46O 486.4311 118.0859
betainylated FS, SE, SG, and ASG were 35 V, 13 V, 10 V, and 15 V, 2 (I.S.) Cholestanol C27H48O 488.4468 118.0859
3 Campesterol C28H48O 500.4468 118.0859
respectively. 4 Stigmasterol C29H48O 512.4468 118.0859
5 Sitosterol C29H50O 514.4624 118.0859
Data analysis 6 (I.S.) Stigmastanol C29H52O 516.4781 118.0859
All sterol lipid classes were quantified by targeted Q-TOF MS/
I.S., internal standard.
MS analysis. Data were processed using Agilent MassHunter qual- a
Stigmasterol and isofucosterol were not distinguished by Q-TOF
itative analysis software. A window of 100 ppm was set for frag- measurements.
ment identification. Data obtained by Agilent MassHunter b
Formula of the parental, non-charged molecule.
qualitative analysis software were further processed using Micro- c
Molecular weight of the cation formed of the parental molecule
soft Excel 2007. Free sterols were quantified using the peak size after derivatization with N-chlorobetainyl chloride.

1042 Journal of Lipid Research Volume 52, 2011

 TABLE 2. Profiling of sterol esters from Arabidopsis and by Q-TOF MS/MS. From this experiment, a correction fac-
Q7

+ tor for unsaturated sterols was calculated (1.61 ± 0.06; n = 7). SEs
Compound Sterol ester (M + NH4) Product ion FS
number molecular species
a
Formula (M)b (m/z)
c
(m/z) were quantified by the formation of sterol-specific fragments (FS)
(Fig. 1). Two pairs of internal standards were used to compensate
7 16:3-Cholesterol C43H70O2 636.5714 369.3505 for the differences in ion suppression for SEs with saturated or
8 16:2-Cholesterol C43H72O2 638.5871 369.3505
9 (I.S.) 16:1-Cholesterol C43H74O2 640.6027 369.3505 unsaturated acyl groups (Fig. 2). SGs show different fragmenta-
10 (I.S.) 16:0-Cholesterol C43H76O2 642.6184 369.3505 tion patterns depending on their degree of unsaturation of the
11 16:3-Campesterol C44H72O2 650.5871 383.3662 sterol moiety (see Fig. 4). Therefore, a mixture of unsaturated
12 16:2-Campesterol C44H74O2 652.6027 383.3662 and saturated SGs was measured via GC and Q-TOF MS/MS. Af-
13 16:1-Campesterol C44H76O2 654.6184 383.3662 ter normalization to the same molar quantity, the Q-TOF MS/MS
14 16:0-Campesterol C44H78O2 656.6340 383.3662
FS peak intensity was found to be reproducibly smaller for satu-
15 16:3-Stigmasterol C45H72O2 662.5871 395.3662
16 16:2-Stigmasterol C45H74O2 664.6027 395.3662 rated SG than for unsaturated SG by a factor of 0.169 ± 0.017
17 16:3-Sitosterol C45H74O2 664.6027 397.3818 (n = 3). This factor was used for the correction during Q-TOF MS/
18 18:3-Cholesterol C45H74O2 664.6027 369.3505 MS measurements of unsaturated SGs. ASGs with an unsaturated
19 16:1-Stigmasterol C45H76O2 666.6184 395.3662 sterol moiety showed a different fragmentation pattern with a
20 16:2-Sitosterol C45H76O2 666.6184 397.3818 high FS peak, while ASGs with a saturated sterol group showed a
21 18:2-Cholesterol C45H76O2 666.6184 369.3505
22 16:0-Stigmasterol C45H78O2 668.6340 395.3662 high acyl-glucoside-specific fragment (FAG) (see Fig. 4). Quantifi-
23 16:1-Sitosterol C45H78O2 668.6340 397.3818 cation of the FS and FAG fragment peaks in a mixture of soybean
24 (I.S.) 18:1-Cholesterol C45H78O2 668.6340 369.3505 ASGs (containing mostly unsaturated sterols) and hydrogenated
25 16:0-Sitosterol C45H80O2 670.6497 397.3818 ASGs (containing saturated sterols) by Q-TOF MS/MS revealed
26 (I.S.) 18:0-Cholesterol C45H80O2 670.6497 369.3505 that the signal responses for ASGs with unsaturated and saturated
27 18:3-Campesterol C46H76O2 678.6184 383.3662
28 18:2-Campesterol C46H78O2 680.6340 383.3662
sterol moieties were comparable, and, therefore, no correction
29 18:1-Campesterol C46H80O2 682.6497 383.3662 factor was required. Exact masses of all molecular sterol species
30 18:0-Campesterol C46H82O2 684.6653 383.3662 and isotopic distribution of the MS/MS fragments derived from

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31 18:3-Stigmasterol C47H76O2 690.6184 395.3662 the presence of 13C isotopes were calculated using the Agilent
32 18:3-Sitosterol C47H78O2 692.6340 397.3818 MassHunter mass calculator and the Isotope Distribution Calcu-
33 18:2-Stigmasterol C47H78O2 692.6340 395.3662
lator tools. Targeted lists for the molecular species of all sterol
34 20:3-Cholesterol C47H78O2 692.6340 369.3505
35 18:2-Sitosterol C47H80O2 694.6497 397.3818 classes are displayed in Tables 1–4. Isotopic correction was ap-
Q8

36 18:1-Stigmasterol C47H80O2 694.6497 395.3662 plied in all cases where a molecular species exists that contains
37 20:2-Cholesterol C47H80O2 694.6497 369.3505 one additional double bond (i.e., m/z M-2) in the sterol moiety or
38 18:0-Stigmasterol C47H82O2 696.6653 395.3662 in the acyl chain, as outlined in a previous study (45). A linear
39 18:1-Sitosterol C47H82O2 696.6653 397.3818 regression line was calculated for the two pairs of standards used
40 20:1-Cholesterol C47H82O2 696.6653 369.3505
41 18:0-Sitosterol C47H84O2 698.6810 397.3818 for each lipid class to account for the dependence of ionization
42 20:0-Cholesterol C47H84O2 698.6810 369.3505 on the size (m/z) of the parental ion (46). The contents of molec-
43 20:3-Campesterol C48H80O2 706.6497 383.3662 ular species were calculated in µmol g21 FW.
44 20:2-Campesterol C48H82O2 708.6653 383.3662
45 20:1-Campesterol C48H84O2 710.6810 383.3662
46 20:0-Campesterol C48H86O2 712.6966 383.3662
47 20:3-Stigmasterol C49H80O2 718.6497 395.3662 RESULTS
48 22:3-Cholesterol C49H82O2 720.5245 369.3505
49 20:2-Stigmasterol C49H82O2 720.6653 395.3662 Measurement of sterol lipids by Q-TOF MS/MS
50 20:3-Sitosterol C49H82O2 720.6653 397.3818
51 20:1-Stigmasterol C49H84O2 722.6810 395.3662 Direct infusion MS/MS experiments using a triple-
52 20:2-Sitosterol C49H84O2 722.6810 397.3818 quadrupole or a Q-TOF mass spectrometer represent a
53 22:2-Cholesterol C49H84O2 722.6810 369.3505
54 20:0-Stigmasterol C49H86O2 724.6966 395.3662
robust and reliable method for the identification and quanti-
55 20:1-Sitosterol C49H86O2 724.6966 397.3818 fication of complex lipids (26, 28, 46, 47). This strategy has
56 22:1-Cholesterol C49H86O2 724.6966 369.3505 been successfully applied to the measurements of phos-
57 20:0-Sitosterol C49H88O2 726.7123 397.3818
58 22:0-Cholesterol C49H88O2 726.7123 369.3505
phoglycerolipids, glycoglycerolipids, sphingolipids, and
59 22:3-Campesterol C50H84O2 734.5401 383.3662 triacylglycerols in plants, animals, and yeast (28). A major
60 22:2-Campesterol C50H86O2 736.6966 383.3662 drawback of this approach can originate from differences
61 22:1-Campesterol C50H88O2 738.7123 383.3662 in the ionization efficiency of molecules from a complex
62 22:0-Campesterol C50H90O2 740.7279 383.3662
63 22:3-Stigmasterol C51H84O2 746.5401 395.3662 matrix. It is well known that the polarity of a molecule is a
64 22:3-Sitosterol C51H86O2 748.5558 397.3818 major determinant for the ionization efficiency in the ion
65 22:2-Stigmasterol C51H86O2 748.6966 395.3662 source. Therefore, nonpolar molecules can suffer from se-
66 22:1-Stigmasterol C51H88O2 750.7123 395.3662
67 22:2-Sitosterol C51H88O2 750.7123 397.3818 vere ion suppression during ionization. The four sterol
68 22:0-Stigmasterol C51H90O2 752.7279 395.3662 lipid classes present in plants strongly differ in their polari-
69 22:1-Sitosterol C51H90O2 752.7279 397.3818 ties, with FS and SE representing nonpolar lipids, while SG
70 22:0-Sitosterol C51H92O2 754.7436 397.3818
and ASG are polar lipids. To study the influence of ion
I.S., internal standard. suppression, a dilution series was generated from a crude
a
Stigmasterol and isofucosterol were not distinguished by Q-TOF Arabidopsis leaf lipid extract, and peak signals for sterol lip-
measurements.
b
Formula of the parental, non-charged molecule. ids were observed in the MS and MS/MS modes of the
c
Molecular weight of the cation formed of the parental molecule nanospray Q-TOF mass spectrometer. Strong ion suppres-
with ammonium.
sion was observed for FS and SE, while SG and ASG were
less affected (data not shown). Therefore, the lipid extract

Sterol lipid quantification in plants by Q-TOF MS 1043

 TABLE 3. Profiling of sterol glucosides from Arabidopsis
Sterol glucoside molecular
Compound number speciesa
71 Glc-Cholesterol
72 (I.S.) Glc-Cholestanol
73 Glc-Campesterol
74 Glc-Stigmasterol
75 Glc-Sitosterol
76 (I.S.) Glc-Stigmastanol
I.S., internal standard.
a
Stigmasterol and isofucosterol were not distinguished by Q-TOF measurements.
b
Formula of the parental, non-charged molecule.
c
Molecular weight of the cation formed of the parental molecule with ammonium.
was fractionated via SPE on silica columns, and two frac-
tions containing nonpolar lipids (FS and SE) or polar lip-
ids (SG and ASG) were harvested (Fig. 1). This additional
purification step resulted in a strong improvement of ion-
ization efficiency for all sterol lipid classes, in particular
for nonpolar sterol lipids, and was therefore employed for
all further analyses.
Infusion of lipids in methanol-chloroform containing
aqueous ammonium acetate resulted in the ionization of
most sterol lipids (SE, SG, and ASG) as ammonium ad-
ducts (Fig. 2). Free sterols, however, were poorly ionized,
Q9
and, upon fragmentation, many small fragments were pro-
duced, whose number and size depended on the structure
of the respective sterol. Derivatization of target molecules
has been shown to improve ionization and, furthermore,
during fragmentation can yield a strong leaving group,
which can be selected in a precursor ion or neutral loss
scanning experiment. Free cholesterol, dehydrocholes-
terol, and oxysterols were previously quantified in MS/MS
experiments after derivatization to esters of mono-
(dimethylaminoethyl)-succinate, N,N-dimethylglycine, pi-
colinate, or acetate or to hydrazones (30–33). Derivatization
of FSs from yeast with acetylchloride resulted in the syn-
thesis of sterol acetates that were detected as sterol ions
after fragmentation (28). Acetylation of FSs from Arabidop-
sis leaves resulted in high background signals (data not
shown). Derivatization with N-chlorobetainyl chloride has
previously been shown to improve ionization of diacylglyc-
erol in MS/MS experiments (43). Therefore, FSs from
Arabidopsis leaves were betainylated with N-chlorobetainyl
chloride, thereby adding a permanent positive charge to
the molecule. Betainylated sterols were easily ionized with-
out further adduct formation and yielded a characteristic
cation fragment for the betainyl group after collision in-
duced fragmentation (Fig. 3).
Sterol quantification with internal standards
For each lipid class, at least two internal standards were
used for quantification to correct for the differences in sig-
nal intensity, which has been shown to be dependent on
the mass of the parental ion (26, 46). Saturated sterols are
absent from plants and can therefore serve as suitable in-
ternal standards. Thus, cholestanol and stigmastanol were
used as internal standards for free sterol measurements.
Because of their low polarity, SEs are susceptible to ion
suppression, in particular SEs carrying a saturated fatty
1044 Journal of Lipid Research Volume 52, 2011
Formula (M)b (M+NH4)+(m/z)c Product ion FS(m/z)
C33H56O6 566.4415 369.3505
C33H58O6 568.4572 371.3662
C34H58O6 580.4572 383.3662
C35H58O6 592.4572 395.3662
C35H60O6 594.4728 397.3818
C35H62O6 596.4885 399.3975
acid residue, while SEs with unsaturated fatty acids are less
affected. Previously, liquid chromatography MS analysis
with a quadrupole ion trap instrument revealed that signal
intensities of SEs were increased depending on the degree
of saturation of the acyl group (48). Direct infusion nano-
spray MS/MS with a Q-TOF instrument showed that
the signal intensities for SEs carrying mono-, di-, and triun-
saturated acyl groups were comparable. To compensate
for partial ion suppression of SEs with saturated acyl
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groups, two pairs of internal standards were added.
The pairs of 16:0-cholesterol/18:0-cholesterol and 16:1-
cholesterol/18:1-cholesterol were employed for the mea-
surement of SEs with saturated and unsaturated fatty acids,
respectively. For the quantification of SGs, cholestanol-Glc
and stigmastanol-Glc were chemically synthesized. For
ASGs, a commercially available ASG fraction from soybean
was hydrogenated. Two fully saturated ASGs, 16:0-Glc-stig-
mastanol and 18:0-Glc-stigmastanol, were then used as in-
ternal standards for ASG quantification.
Fragmentation patterns of sterol lipids
The basis for lipid quantification via direct infusion MS/
MS is the selection of the parental ion in the first quadru-
pole, the fragmentation in the collision cell, and the iden-
tification and quantification of a characteristic fragment
peak in the last quadrupole (for triple-quadrupole mass
spectrometers) or in the TOF mass analyzer (for Q-TOF).
The fragmentor voltage was optimized to avoid premature
fragmentation of conjugated sterols before reaching the
collision cell. Furthermore, collision energies were opti-
mized for maximal signal intensities for the desired frag-
ments. The selected fragment ions are shown in Figs. 3
and 4.
Betainylated FSs carry a permanent positive charge that
renders adduct formation unnecessary. Fragmentation re-
sulted in the neutral loss of the sterol backbone (Fig. 3,
shown as NS) and in the detection of the characteristic be-
tainyl cation at m/z of 118.0859. Fragmentation of ammo-
nium adducts of SEs led to the neutral loss of the respective
acyl groups (Fig. 3). The signal of the sterol-specific back-
bone fragment (FS) was used for quantification.
Similarly, fragmentation of SG produced an FS peak cor-
Q10
responding to that of the sterol backbone (Fig. 4). Com-
parison of signal intensities obtained in the MS mode with
those obtained in the MS/MS mode confirmed that frag-
mentation of unsaturated SGs yields a high peak for the FS
 TABLE 4. Profiling of acylated sterol glucosides from Arabidopsis

Compound number Acylated sterol glucoside molecular speciesa Formula (M)b (M+NH4)+(m/z)c Product ion FS (m/z) Product ion FAG (m/z)

77 16:3-Glc-Cholesterol C49H80O7 798.6242 369.3505 395.2428

78 16:2 Glc-Cholesterol C49H82O7 800.6399 369.3505 397.2585
79 16:1-Glc-Cholesterol C49H84O7 802.6555 369.3505 399.2741
80 16:0-Glc-Cholesterol C49H86O7 804.6712 369.3505 401.2898
81 16:3-Glc-Campesterol C50H82O7 812.6399 383.3662 395.2428
82 16:2-Glc-Campesterol C50H84O7 814.6555 383.3662 397.2585
83 16:1-Glc-Campesterol C50H86O7 816.6712 383.3662 399.2741
84 16:0-Glc-Campesterol C50H88O7 818.6868 383.3662 401.2898
85 (I.S.) 16:0-Glc-Campestanol C50H90O7 820.7025 385.3818 401.2898
86 16:3-Glc-Stigmasterol C51H82O7 824.6399 395.3662 395.2428
87 16:2-Glc-Stigmasterol C51H84O7 826.6555 395.3662 397.2585
88 16:3-Glc-Sitosterol C51H84O7 826.6555 397.3818 395.2428
89 18:3-Glc-Cholesterol C51H84O7 826.6555 369.3505 423.2741
90 16:1-Glc-Stigmasterol C51H86O7 828.6712 395.3662 399.2741
91 16:2-Glc-Sitosterol C51H86O7 828.6712 397.3818 397.2585
92 18:2 Glc-Cholesterol C51H86O7 828.6712 369.3505 425.2898
93 16:0-Glc-Stigmasterol C51H88O7 830.6868 395.3662 401.2898
94 16:1-Glc-Sitosterol C51H88O7 830.6868 397.3818 399.2741
95 18:1-Glc-Cholesterol C51H88O7 830.6868 369.3505 427.3054
96 16:0-Glc-Sitosterol C51H90O7 832.7025 397.3818 401.2898
97 18:0-Glc-Cholesterol C51H90O7 832.7025 369.3505 429.3211
98 (I.S.) 16:0-Glc-Stigmastanol C51H92O7 834.7181 399.3975 401.2898
99 18:3-Glc-Campesterol C52H86O7 840.6712 383.3662 423.2741
100 18:2-Glc-Campesterol C52H88O7 842.6868 383.3662 425.2898
101 18:1-Glc-Campesterol C52H90O7 844.7025 383.3662 427.3054

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102 18:0-Glc-Campesterol C52H92O7 846.7181 383.3662 429.3211
103 (I.S.) 18:0-Glc-Campestanol C52H94O7 848.7338 385.3818 429.3211
104 18:3-Glc-Stigmasterol C53H86O7 852.6712 395.3662 423.2741
105 18:3-Glc-Sitosterol C53H88O7 854.6868 397.3818 423.2741
106 18:2-Glc-Stigmasterol C53H88O7 854.6868 395.3662 425.2898
107 20:3-Glc-Cholesterol C53H88O7 854.6868 369.3505 451.3054
108 18:2-Glc-Sitosterol C53H90O7 856.7025 397.3818 425.2898
109 18:1-Glc-Stigmasterol C53H90O7 856.7025 395.3662 427.3054
110 20:2-Glc-Cholesterol C53H90O7 856.7025 369.3505 453.3211
111 18:0-Glc-Stigmasterol C53H92O7 858.7181 395.3662 429.3211
112 18:1-Glc-Sitosterol C53H92O7 858.7181 397.3818 427.3054
113 20:1-Glc-Cholesterol C53H92O7 858.7181 369.3505 455.3367
114 18:0-Glc-Sitosterol C53H94O7 860.7338 397.3818 429.3211
115 20:0-Glc-Cholesterol C53H94O7 860.7338 369.3505 457.3524
116 (I.S.) 18:0-Glc-Stigmastanol C53H96O7 862.7494 399.3975 429.3211
117 20:3-Glc-Campesterol C54H90O7 868.7025 383.3662 451.3054
118 20:2-Glc-Campesterol C54H92O7 870.7181 383.3662 453.3211
119 20:1-Glc-Campesterol C54H94O7 872.7338 383.3662 455.3367
120 20:0-Glc-Campesterol C54H96O7 874.7494 383.3662 457.3524
121 20:3-Glc-Stigmasterol C55H90O7 880.7025 395.3662 451.3054
123 22:3-Glc-Cholesterol C55H92O7 882.5773 369.3505 479.3367
124 20:2-Glc-Stigmasterol C55H92O7 882.7181 395.3662 453.3211
125 20:3-Glc-Sitosterol C55H92O7 882.7181 397.3818 451.3054
126 20:1-Glc-Stigmasterol C55H94O7 884.7338 395.3662 455.3367
127 20:2-Glc-Sitosterol C55H94O7 884.7338 397.3818 453.3211
128 22:2-Glc-Cholesterol C55H94O7 884.7338 369.3505 481.3524
129 20:0-Glc-Stigmasterol C55H96O7 886.7494 395.3662 457.3524
130 20:1-Glc-Sitosterol C55H96O7 886.7494 397.3818 455.3367
131 22:1-Glc-Cholesterol C55H96O7 886.7494 369.3505 483.3680
132 20:0-Glc-Sitosterol C55H98O7 888.7651 397.3818 457.3524
133 22:0-Glc-Cholesterol C55H98O7 888.7651 369.3505 485.3837
134 22:3-Glc-Campesterol C56H94O7 896.5929 383.3662 479.3367
135 22:2-Glc-Campesterol C56H96O7 898.7494 383.3662 481.3524
136 22:1-Glc-Campesterol C56H98O7 900.7651 383.3662 483.3680
137 22:0-Glc-Campesterol C56H100O7 902.7807 383.3662 485.3837
138 (I.S.) 22:0-Glc-Campestanol C56H102O7 904.7964 385.3818 485.3837
139 22:3-Glc-Stigmasterol C57H94O7 908.5929 395.3662 479.3367
140 22:3-Glc-Sitosterol C57H96O7 910.6086 397.3818 479.3367
141 22:2-Glc-Stigmasterol C57H96O7 910.7494 395.3662 481.3524
142 22:1-Glc-Stigmasterol C57H98O7 912.7651 395.3662 483.3680
143 22:2-Glc-Sitosterol C57H98O7 912.7651 397.3818 481.3524
144 22:0-Glc-Stigmasterol C57H100O7 914.7807 395.3662 485.3837
145 22:1-Glc-Sitosterol C57H100O7 914.7807 397.3818 483.3680
146 22:0-Glc-Sitosterol C57H102O7 916.7964 397.3818 485.3837
147 (I.S.) 22:0-Glc-Stigmastanol C57H104O7 918.8120 399.3975 485.3837

I.S., internal standard.

a
Stigmasterol and isofucosterol were not distinguished by Q-TOF measurements.
b
Formula of the parental, non-charged molecule.
c
Molecular weight of the cation formed of the parental molecule with ammonium.

Sterol lipid quantification in plants by Q-TOF MS 1045

 Downloaded from www.jlr.org by guest, on December 9, 2020
Fig. 3. Fragmentation pattern of free sterols and sterol esters. A: Q-TOF MS/MS spectra of betainylated FSs with the corresponding frag-
mentation patterns and calculated masses. Fragmentation of the betainyl derivatives of sitosterol, stigmasterol, campesterol, and stigmas-
tanol (internal standard) results in the neutral loss of the sterol moiety along with the accumulation of the betainyl fragment FB at m/z of
118.0859. B: Q-TOF MS/MS spectra of SEs. Fragmentation of the ammonium adducts of SEs leads to the release of the corresponding
sterol cation FS. 16:1-cholesterol was used as the internal standard.

(Fig. 4). For saturated SGs, the FS peak was much smaller,
and a number of additional peaks occurred at lower m/z.
This was of particular importance because the internal
standards contained saturated sterol moieties, while plant
SGs contain mostly unsaturated sterols. Application of cor-
rection factors has previously been described for the quan-
tification of sphingolipid classes (3). A similar approach
was therefore followed for the quantification of SGs by cal-
culation of a correction factor to compensate for the lower
FS signal intensities of saturated versus unsaturated SGs.
Similar to SGs, the fragmentation pattern of the ASGs
was different when one or more double bonds were pre-
sent in the sterol moiety (Fig. 4). The fragmentation of
ASGs carrying unsaturated sterol moieties resulted in the
accumulation of a large peak for the sterol fragment FS
(Fig. 4). Fragmentation of ASGs with a saturated sterol
backbone, however, led to the formation of a large peak
for the FAG (Fig. 4). Therefore, the FS and FAG fragment
peaks were employed for quantification of ASGs with un-
saturated or saturated sterol moieties, respectively.
The linearity of signal intensities was evaluated for each
sterol lipid class, using the corresponding standards. To
compensate for fluctuations in the nanospray flow of the
ion source, the signal of each sterol lipid standard was nor-
malized to a second internal standard of the same lipid
class, which was kept at constant concentration. The stan-
dard curves of the normalized, logarithmic signals were
then plotted relative to the logarithmic amount of the var-
iable standard (Fig. 5). Standard curves showed that the
signals were linear over a range of 3 to 4 orders of magni-
tude for FS, SE, and SG and greater than 2 orders of mag-
nitude for ASG. Correlation coefficients for linearity for
all four lipid classes were calculated to be higher than
0.99.
Scanning for molecular species composition of sterol
lipids in Arabidopsis leaves
Sitosterol and campesterol were previously shown to
be the most abundant sterols in Arabidopsis (15, 49).
Cholesterol, stigmasterol, and isofucosterol are minor

1046 Journal of Lipid Research Volume 52, 2011

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Fig. 4. Fragmentation pattern of sterol glucosides and acylated sterol glucosides. A: Q-TOF MS/MS spectra of different SGs. Collision
induced dissociation of the ammonium adduct of SGs produces a sterol fragment FS. The spectrum of the saturated stigmastanol-Glc (used
as the internal standard) reveals a peak of lower intensity corresponding to the sterol fragment Fs and the presence of additional peaks. B:
Q-TOF MS/MS spectra of ASGs. Note that fragmentation of ASGs with unsaturated sterol moieties leads to the release of the sterol cation
FS. Fragmentation of ASGs with saturated sterol moieties (16:0-Glc-stigmastanol, internal standard) leads to the accumulation of a peak at
401.2898, corresponding to the 16:0-Glc cation.

components. Nontargeted screening for molecular spe-
cies of sterol lipids using the Q-TOF MS and MS/MS
modes confirmed this result (data not shown). There-
fore, these sterols were selected for a targeted approach
to quantify the different sterol lipid classes. As stigmas-
terol and isofucosterol are isomeric and, therefore, con-
jugated sterol lipids containing either of these two sterols
yield isobaric fragments after collision induced dissocia-
tion, they were quantified together. For SE and ASG, a
large number of molecular species carrying different
acyl groups were detected by MS and MS/MS experiments,
with acyl groups ranging from 16 to 22 carbon atoms and
containing 0 to 3 double bonds. While not all of these
acyl groups were found in SEs or ASGs of Arabidopsis, a
wide range of molecular species was selected for targeted
MS/MS analysis to cover possible alterations in the fatty
acid pattern during stress or in different plant species
(Fig. 6). The most abundant SEs in Arabidopsis leaves
were 18:3-sitosterol and 18:2-sitosterol. Interestingly, a
considerable amount of SEs contained very-long-chain
fatty acids, e.g., 20:0-cholesterol and 20:0-sitosterol. The
molecular species pattern of ASGs was dominated by
16:0-Glc-sitosterol, with smaller amounts of 18:3-Glc-
sitosterol and 18:2-Glc-sitosterol (Fig. 6). Targeted lists
including the masses of all sterol lipid classes for paren-
tal ions and the fragment ions used for quantification
are displayed in Tables 1–4.
Comparison of sterol lipid quantification by Q-TOF
MS/MS and TLC/GC
TLC/GC quantification of sterol lipids from Arabidopsis
leaves was used to validate the quantification results ac-
quired by Q-TOF MS/MS. To this end, a total lipid extract
from Arabidopsis leaves including internal standards was

Sterol lipid quantification in plants by Q-TOF MS 1047


Fig. 5. Standard curves for sterol lipids. Different amounts of
each lipid standard were infused into the Q-TOF mass spectrome-
ter in the presence of a constant amount of an internal standard,
and the ratio of the MS/MS signals was plotted against the variable
standard content (in nmol per 200 µl). The data show the means ±
SD of at least three experiments and regression lines. Axes show
logarithmic scaling. A: Standard curve for stigmastanol normalized
to cholestanol. B: Standard curve for 18:1-cholesterol normalized
to 16:1-cholesterol. C: Standard curve for stigmastanol-Glc normal-
ized to cholestanol-Glc. D: Standard curve for acylated stigmas-
tanol-glucoside normalized to acylated sitosterol-glucoside.
fractionated into nonpolar (FS and SE) and polar (SG and
ASG) sterols via SPE on silica columns. One aliquot was
taken for Q-TOF analysis. A second aliquot was used for
further separation by SPE and TLC and quantification by
GC after hydrolysis and silylation (Fig. 1). Figure 7 shows a
comparison of the sterol lipid contents as measured by
TLC/GC and Q-TOF MS/MS. The two methods resulted
in the same overall composition and comparable absolute
amounts of sterol lipids from Arabidopsis leaves. FSs repre-
sent the predominant sterol lipid class, while SEs and SGs
1048 Journal of Lipid Research Volume 52, 2011
are less abundant, and ASGs represent a minor lipid
class.
Changes in sterol lipid composition during phosphate
deprivation
Phosphate deficiency represents one of the most se-
vere stimuli for alterations of membrane lipid composi-
tion in plants. While the changes in the contents of
phosphoglycerolipids and glycoglycerolipids during
phosphate deficiency have been well documented, the
effects on the amounts of sterol lipids are less clear.
Therefore, the content and composition of sterol lipids
were measured by Q-TOF MS/MS after phosphate depri-
vation. Lipids were isolated from leaves or roots of Arabi-
dopsis plants grown on synthetic medium with or without
phosphate. Furthermore, lipids were extracted from the
Arabidopsis pho1 mutant. Leaves of the pho1 mutant are
phosphate-deficient due to a mutation in a phosphate
transporter that affects phosphate transport from the
root to the shoot (38, 50). Figure 8 shows the sterol lipid
content measured by Q-TOF MS/MS in leaves of the wild
type (WT) and the pho1 mutant and in leaves and roots of
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plants grown on synthetic medium in the presence or ab-
sence of phosphate. Phosphate deprivation caused a sig-
nificant (P < 0.02) increase in the amounts of SG and
ASG in leaves, which could also be detected in leaves of
the pho1 mutant, compared with those in the WT. The
amount of SG was also significantly increased in roots
during phosphate deprivation, while the ASG content in
roots was not affected. At the same time, the content of
FS was slightly increased in leaves of phosphate-deprived
plants, but this change was not significant, and FS
remained unchanged in roots. These findings were in
accordance with data previously obtained from phos-
phate-limited oat (22). The amounts of SE were signifi-
cantly increased in phosphate-deprived leaves and
phosphate-deprived roots of the pho1 mutant. Further-
more, leaves of Arabidopsis plants grown on synthetic me-
dium consistently contained a higher SE content than
soil-grown plants, which was further increased during
phosphate deprivation (Fig. 8).
The amounts of cholesterol, campesterol, stigmas-
terol/isofucosterol, and sitosterol in the different sterol
lipid classes were very similar upon phosphate depriva-
tion (Fig. 9). Although some of the changes were calcu-
lated to be significant (P < 0.02), the differences were
minor. Figure 10 shows alterations in fatty acid composi-
tion of SEs and ASGs during phosphate deprivation. The
18:2 content of SEs in the pho1 mutant and in the leaves
of WT plants grown in the absence of phosphate was sig-
nificantly reduced (P < 0.02). At the same time, the
amounts of 18:3 content in SEs were significantly in-
creased. Furthermore, a substantial proportion of saturated
very-long-chain (20:0; 22:0) acyl groups were found in
SEs of WT roots, and the amounts of these acyl groups
were significantly decreased during phosphate depriva-
tion. In leaves, the amounts of 18:2 ASGs were signifi-
cantly decreased during phosphate deprivation with a
concomitant increase in 18:3, similar to the changes in
 Downloaded from www.jlr.org by guest, on December 9, 2020
Fig. 6. Molecular species composition of sterol esters and acylated sterol glucosides determined by Q-TOF MS/MS. A: Sterol esters; (B)
acylated sterol glucosides. Data show means ± SD of four measurements. Stigmasterol and isofucosterol-containing molecular species were
not distinguished as these two sterols are isomeric.

acyl composition of SEs. In roots, 16:0 was significantly
decreased in ASGs with a concomitant accumulation of
18:3, indicating that there was a shift from saturated to
unsaturated acyl groups.
DISCUSSION
The development of high-resolution mass spectrome-
ters has provided the means for the establishment of new
approaches to the identification and quantification of
plant metabolites. In contrast to GC-based methods, which
usually depend on hydrolyzation and derivatization of
complex lipids, nondestructive methods are required for
the direct measurements of membrane and storage lipids.
Here we describe the development of a comprehensive
strategy for the identification and quantification of the
molecular species of all known sterol lipid classes in plants
via Q-TOF MS/MS.
Sterol lipid classes in plants differ with regard to their
polarity, which determines the ionization and fragmenta-
tion characteristics in MS and MS/MS experiments. At-
tempts to measure the molecular species of all four sterol

Sterol lipid quantification in plants by Q-TOF MS 1049

Fig. 7. Comparison of sterol lipid quantification by Q-TOF MS/
MS measurements versus those with TLC/GC. Lipids were isolated
from Arabidopsis leaves in the presence of internal standards and
fractionated by SPE into nonpolar lipids (FS, SE) and polar lipids
(SG, ASG). One aliquot each was analyzed by TLC/GC (black bars)
or by Q-TOF MS/MS (gray bars). Bars show means ± SD of four
measurements.

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lipid classes by direct infusion MS/MS experiments of a
crude lipid extract were not satisfactory. The ionization
efficiency of the different sterol lipids was strongly im-
proved after SPE on silica columns. However, ionization
efficiency of FSs was still inadequate. Conversion of FS into
betainyl derivatives strongly improved ionization and pro-
vided the means for detection of a strong leaving group in
MS/MS. Ionization of SEs was dependent on the presence
or absence of a double bond in the acyl chain. SEs with
saturated acyl chains were more prone to ion suppression
than those containing at least one double bond. For this
reason, the saturated and unsaturated SEs were quantified
relative to the respective saturated or unsaturated internal
SE standards.
While ionization efficiency of SGs and ASGs was high,
their fragmentation patterns were found to depend
Fig. 9. Sterol composition of Arabidopsis leaves and roots mea-
sured by Q-TOF MS/MS. Lipids were extracted from Arabidopsis
leaves of WT and pho1 mutant plants grown on soil and from leaves
and roots of WT plants grown on synthetic medium with or without
phosphate. Bars show the sterol composition of free sterols (A),
sterol esters (B), sterol glucosides (C), and acylated sterol gluco-
sides (D). Data are means ± SD of five replicas. The results were
confirmed by a second independent biological experiment. Sterol
lipids containing stigmasterol or isofucosterol were not distin-
guished. Values that are significantly different from those of the
WT or the phosphate-containing control and are higher than 2
mol% are indicated by an asterisk (according to Student’s t test;
P < 0.02).

Fig. 8. Sterol lipid content in Arabidopsis leaves and roots during
phosphate deprivation measured by Q-TOF MS/MS. Lipids were
extracted from Arabidopsis leaves of WT and pho1 mutant plants
grown on soil and from leaves and roots of WT plants grown on
synthetic medium with or without phosphate. The bars show sterol
lipid content (means ± SD of five measurements). Data were con-
firmed by a second independent biological experiment. Values
significantly different from those of the WT or from the phosphate-
containing control are indicated by an asterisk (according to Stu-
dent’s t test; P < 0.02).
strongly on the presence or absence of a double bond in
the sterol moiety. The peak sizes of the sterol fragments
were reduced for saturated SGs compared with those of
unsaturated SGs. A correction factor was determined to
compensate for the differences in fragmentation effi-
ciency. Similarly, the abundance of the sterol fragment, FS,
of ASGs with saturated sterol moiety was much lower than
that of ASGs with unsaturated sterol. The FS peak of ASGs
with saturated sterol moiety was therefore not suitable for

1050 Journal of Lipid Research Volume 52, 2011

 Downloaded from www.jlr.org by guest, on December 9, 2020
Fig. 10. Fatty acid composition of sterol esters and acylated sterol glucosides determined by Q-TOF MS/
MS. A: Sterol esters; (B) acylated sterol glucosides. Sterol lipids were isolated from leaves of WT and pho1
mutant plants grown on soil and from leaves and roots of WT plants grown on synthetic medium with or
without phosphate. Bars show acyl composition as means ± SD of five measurements. Data were confirmed
by a second independent biological experiment. Values that are significantly different from those of the WT
or phosphate-containing control and higher than 2 mol% are indicated by an asterisk (according to Stu-
dent’s t test; P < 0.02).

quantification, and a different fragment, FAG, correspond-
ing to the acylated glucose head group was used. In the
present study, saturated sterols (internal standards) and
sterols containing one double bond at position 5 of the
ring system (cholesterol, sitosterol, campesterol) or stig-
masterol (one double bond at position 5, one double bond
at position 22) were analyzed (Figs. 3, 4). Signal intensities
for stigmasterol and sitosterol containing sterol lipids were
very similar, indicating that the additional double bond
at position 22 has a minor impact on ionization and
fragmentation.
In agreement with previous studies (15, 49), sitosterol
and campesterol were found to be the predominant sterol
components in Arabidopsis leaves, as determined by Q-TOF
MS/MS, while cholesterol, stigmasterol, and isofucosterol
were minor components. Stigmasterol and isofucosterol
are isomers with the same molecular formula (C29H48O).
Conjugated lipids containing stigmasterol or isofucosterol
give rise to isobaric fragments and therefore cannot be
separated in MS/MS experiments.
Unsaturated acyl groups, mostly 18:2 and 18:3, were
prevalent in SEs of leaves and roots (Fig. 4, 7). Interest-
ingly, SEs from Arabidopsis roots contained a considerable
amount of 22:0, which was absent from leaves. Roots are
known to contain only small amounts of unsaturated fatty
acids. The large amount of 22:0 in root SEs suggests that a
considerable proportion of acyl groups in SEs was derived
from the fatty acid elongation pathway at the endoplasmic
reticulum. In contrast to SEs, saturated long chain fatty
acids (16:0, 18:0) are prevalent in ASGs, equivalent to
40%–50% of all fatty acids of ASGs. In addition, smaller
amounts of 18:1, 18:2, and 18:3 are found in ASGs.
The contents of sterol lipids obtained by Q-TOF MS/MS
quantification were highly similar to the sterol lipid amounts
measured by TLC/GC analysis (Fig. 7). Using a combina-
tion of TLC and GC, the contents of FS and SE in Arabidop-
sis leaves were previously determined as 1,021 and 174 µg
g21 dry weight (DW), respectively (49). This corresponds to
2.4 and 0.26 µmol g21 DW, respectively. Higher values
(ⵑ1,600 and 380 µg g21 DW for FS and SE, respectively)
were measured in a different study (11). Considering that
the DW-to-FW ratio for Arabidopsis leaves is ⵑ0.1, this trans-
lates into ⵑ0.24–0.38 and 0.026–0.056 µmol g21 FW for FS
and SE, respectively, which is in the same range as the values
of 0.33 and 0.036 µmol g21 FW determined by Q-TOF MS/
MS in this study (Fig. 7). The ratios of SG and ASG in Arab-
idopsis leaves relative to the other sterol lipids were previ-
ously calculated using the equations (SG + ASG)/FS = 0.15
and ASG/SE = 0.41 (15). These ratios are comparable to
those calculated from Q-TOF MS/MS measurements, i.e.,
(SG + ASG)/FS = 0.14 and ASG/SE = 0.35 (Fig. 7).
The sterol composition percentages of Arabidopsis leaves
were previously measured as 4, 16, 2, and 78% and 13, 12,
15, and 60% for cholesterol, campesterol, isofucosterol, and

Sterol lipid quantification in plants by Q-TOF MS 1051

sitosterol in FS and SE, respectively (49). These values are in
accordance with those measured by Q-TOF MS/MS, 8, 15,
11, and 67% and 12, 17, 16, and 55% for cholesterol, campes-
terol, stigmasterol/isofucosterol, and sitosterol in FS and
SE, respectively (Fig. 9). As indicated above, stigmasterol
and isofucosterol cannot be separated by Q-TOF MS/MS.
Phosphate deficiency is known to cause a strong increase
in the glycolipids DGDG and SQDG and a concomitant
decrease in phospholipids. This adaptive process provides
the means to save phosphate during growth on phosphate-
limiting soils (2). SG and ASG also represent phosphate-free
glycolipids and are localized to extraplastidial mem-
branes, in particular to the plasma membrane. Previously,
changes in plasma membrane lipid composition were
recorded for oat roots (22, 24). After growth on phos-
phate-deficient medium, there was an increase in the pro-
portion of SG and ASG relative to the total oat root plasma
membrane lipids. Measurements of sterol lipids in Arabi-
dopsis leaves and roots by Q-TOF MS/MS after phosphate
deprivation were in agreement with the results previously
obtained with oat. In leaves of the pho1 mutant and the
WT plants deprived of phosphate, SG and ASG were in-
creased, while in roots, there were only minor changes in
ASG. In addition, the SE content in leaves and roots was
strongly increased during phosphate deprivation. The SE
content was not determined during phosphate depriva-
tion in oat, as it does not represent an authentic plasma
membrane lipid (22, 24). Furthermore, the SE content in
leaves of Arabidopsis plants grown on synthetic medium was
elevated compared with that in plants grown on soil. Previ-
ous studies showed that the amount of SE in plants de-
pends on the developmental stage, light conditions, and
tissue type (51). In general, the amount of SE increases
with plant age and during biotic and abiotic stress includ-
ing senescence. It is believed that the accumulation of SE
in oil bodies of leaf cells is in part due to the acylation of
FS derived from the plasma membrane and thus repre-
sents a stress-related regulatory mechanism of membrane
lipid homeostasis. For this reason, the increase of SE un-
der conditions of phosphate deprivation or after transfer
to synthetic medium presumably represents a general
stress response, rather than a specific process related to
nutrient availability.
The acyl compositions of SE and ASG were more suscep-
tible to changes in phosphate supply than the sterol com-
position, which remained more or less unchanged. An
increase of 18:3 with a concomitant decrease of 18:2 acyl
chains in SE and ASG was observed in the pho1 mutant and
in WT leaves depleted of phosphate. In SEs of roots, the
content of saturated very-long-chain fatty acids (20:0; 22:0)
was decreased. This can be explained by an increased syn-
thesis of SEs with 18:2 and 18:3 acyl chains.
Nanospray direct infusion Q-TOF MS with fragment-
specific MS/MS experiments provides a robust and sensitive
method for the quantification of sterol lipids. Signals for all
sterol lipid classes were found to be linear over greater than
2 (ASG) and 3–4 (FS, SE, SG) orders of magnitude, which
was sufficient to accurately quantify all molecular species of
sterol lipids in Arabidopsis leaves. An important advantage
1052 Journal of Lipid Research Volume 52, 2011
of the Q-TOF method over classical TLC/GC-based proto-
cols is the fact that detailed information on the molecular
species composition of SEs and ASGs can be obtained. Fur-
thermore, this method can easily be extended to measure
sterol lipids in different tissues and in other plant species
or organisms by adding further sterol-specific molecular
species to the targeted MS/MS lists.
Nanospray ion sources are known to provide a strong
increase in sensitivity compared with ESI. This is due in
part to the fact that ionization efficiency is improved be-
cause of the reduced sizes of solvent droplets released dur-
ing infusion and to the fact that a lower sample volume is
required. In fact, all sterol lipid measurements by Q-TOF
MS/MS could be done with a lipid extract derived from a
single Arabidopsis leaf. The lower limit for accurate quanti-
fication of the molecular species of sterol lipids (Figs. 4
and 5) was in the range of 0.01 nmol in 200 µl of solvent by
21
Q-TOF MS/MS. At a flow rate of 1 µl min , a measuring
time of 5 min (equivalent to 5 µl) usually is sufficient to
perform the fragmentation experiments required for
quantification of all sterol lipids. Therefore, the lower limit
for quantification of molecular sterol lipid species is around
Downloaded from www.jlr.org by guest, on December 9, 2020
0.5 pmol. In comparison, the minimal amount of sterol
lipid required for quantification by TLC and GC or by
HPLC with an evaporative light-scattering detector lies in
the low microgram (i.e., low nanomolar) range. Further-
more, the minimal amount of tissue required for Q-TOF
MS/MS measurements of all four sterol lipid classes is only
20 mg compared with ⵑ2 g required for TLC/GC quanti-
fication. Therefore, the Q-TOF-based method is far more
Q11
sensitive than “classic” approaches of sterol lipid measure-
ments. A further advantage of the Q-TOF method is the
fact that it is much less labor intensive as it only requires
SPE separation but is independent of TLC.
The sterol lipid quantification method was developed
on a Q-TOF instrument equipped with a direct infusion
nanospray ion source. Quantification was based on MS/
MS experiments. The signal intensities for the characteris-
tic fragments were electronically extracted from the spec-
tra of all parental ions simulating neutral loss scanning
experiments (e.g., for SG) or precursor ion scanning ex-
periments (e.g., for betainylated FS). For this reason, the
Q-TOF strategy of sterol lipid quantification can easily be
transferred to other tandem mass spectrometers including
triple-quadrupole instruments equipped with a nanospray
or ESI source.
The authors thank Helga Peisker (University of Bonn) for
technical assistance during Q-TOF measurements and Felix
Lippold (University of Bonn) for support with data analysis.
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